Mutations in transmembrane channel-like gene 1 (TMC1) underlie dominant, progressive hearing loss (DFNA36) and recessive nonsyndromic sensorineural hearing loss (DFNB7/B11) in humans (Kurima et al., 2002). Similarly, semidominant and recessive alleles of Tmc1 cause hearing loss in Beethoven (Bth) and deafness (dn) mutant mice (Vreugde et al.,2002; Kurima et al., 2002). Tmc1 is a member of the Tmc gene family that includes seven other paralogs in mammals (Keresztes et al., 2003). Tmc1 and closely related Tmc2 are expressed in auditory and vestibular hair cells of the mouse inner ear. We have recently demonstrated that mice that lack Tmc1 and Tmc2 are deaf and suffer profound vestibular dysfunction. The data suggest that Tmc1 and Tmc2 are essential for normal hair cell function but their precise molecular function remains elusive. TMC1 and TMC2 proteins may be components of the cell body required for folding or trafficking of transduction molecules, they may function as linker proteins, mechanically in series with the mechanotransduction channel or they may be pore-forming subunits of the hair cell transduction channel itself. To investigate possible TMC functions we have designed a systematic and comprehensive research strategy that will allow us to distinguish amongst these various hypotheses. We will begin with a thorough biophysical characterization of the properties of hair cell transduction in mutant mice that express either Tmc1 or Tmc2 alone. Next, we will introduce mutant and chimeric forms of Tmc1 and/or Tmc2 into hair cells that lack both genes. Our previous work (Kawashima et al., 2011) demonstrated rescue of mechanotransduction in hair cells of Tmc1/Tmc2 knockout mice transfected with wild-type Tmc1 or Tmc2. Therefore, we predict that similar experiments with mutant Tmc genes will provide a novel platform for structure/function studies in native hair cells which will allow us to identify the function of TMC proteins. Lastly, e will localize TMC proteins in hair cells of Tmc1/Tmc2 knockout mice transfected with wild-type and mutant TMC constructs fused to short polypeptide epitope tags. Immunolocalization with antibodies specific to the epitope tag will provide localization data superior to that currently available. Taken together, the data that emerge from the proposed studies will help identify the function of TMC proteins in hair cells and may support their role as components of the hair cell transduction apparatus.